This project focuses on molecular properties, regulation, and functional roles of ion channels in T lymphocytes. Using patch-clamp techniques, we have characterized a diverse set of functionally significant Ca2+, K+, and CI ion channels in human and mouse T cells. These channels are differentially expressed depending on the developmental and activation state and have been shown to contribute to T-cell receptor signaling leading to gene expression, secretion of lymphokines, and cell proliferation. Ion channels in the immune system offer promising targets for development of therapeutic agents for immunomodulation, based upon specific channel blockade. Using single cell patch-clamp and [Ca2+]j imaging techniques, together with molecular and biochemical approaches, this grant renewal application will focus on the two types of Ca2+ channel found in T lymphocytes. Ca2+ release-activated Ca2+ (CRAG) channels are opened upon depletion of intracellular Ca2+ stores and are crucial for sustained [Ca2+]i signaling that leads to gene expression in T cells. We recently discovered Stim and STIM1 as essential and conserved components of CRAG channel function in Drosophila and human cells. In Specific Aim 1, we will use RNAi to screen for additional components of the CRAG channel, define the mechanism of STIM1 in CRAG channel activation, and investigate the role of STIM1 in functional T cell responses. It is now clear that the native MIC current in T cells represents tetramers of TRPM7, a protein with functional channel and kinase domains. MIC channel expression is up-regulated during the activation of T cells to proliferate. In Specific Aim 2 we will identify the molecular basis for MIC channel gating, define mechanisms for ion permeation and block, and determine the functional role of MIC channels in T cell activation. Through the proposed experiments in this renewal application, we seek a clearer understanding how CRAG and MIC channels function as ion channels and how they regulate Ca2+ influx in T cells and corresponding cell functions that underlie the immune response.